The goal of this project is to develop modulators of radiation response able to overcome radiation resistance in human prostate tumor cells. Although radiation is able of permanently eradicate localized human prostate tumors, nearly 30% of patients treated with potentially curative radiotherapy relapse at the sites of the irradiated tumor due to residual radiation-resistant clonogens. In this application we propose to explore the mechanisms of resistance to radiation-induced apoptosis for the highly resistant, well-characterized human prostate cancer cell lines-LNCaP and CRW22Rv 1, Our preliminary data Show that activation of protein kinase C (PKC) isoforms by the phorbol ester 12,O-tetradecanoyl phorbol-13-acetate, (TPA), induces a moderate apoptotic response in these tumor cells, and sensitizes them towards radiation, induced apoptosis. We also showed that this apoptotic response is mediated by the enzyme ceramide synthase (CS) and de novo synthesis of ceramide. CS activity was previously reported by us to be negatively regulated by the Ataxia Telangiectasia-Mutated (ATM) gene. Our preliminary studies related to this application showed that in LNCaP and CRW22Rvl cells, TPA reduces ATM levels. We observed a similar reduction in ATM protein levels and radiosensitization using a diacylgycerol (DAG)-lactone, HK654, or antisense oligonucleotides to ATM (AS-ATM-ODNs). In this project, we propose to define the involvement of PKCa and PKC5 in TPAinduced apoptosis via activation of the ATM-CS pathway. We will also study the mechanisms of CS involvement in mitochondrial apoptosis, ceramide generation within the mitochondria, and the interaction between ceramide, PKC6 and/or Bax. Finally, we propose to study the role of the ATM-CS pathway in vivo in LNCaP and CWR22Rvl tumors growing orthotopically in nude mice. We will study the response to TPA ( radiation in response to both single-dose radiation as well as to fractionated radiation and will correlate tumor growth in these animals with tissue readouts. The proposed Specific Aims are interactive and address new and heretofore unknown mechanisms of TPA(radiation-induced cell death in prostate cancer cells and prostate animal models. Our research plan presents a signaling-based approach, to investigate new I hypotheses, about mechanisms of radiation resistance, which could provide a basis for modulation of the l radiation response in resistant human prostate tumor clones, with potential for clinical applications in the radiation management of human prostate cancer.